scRNA-seq reveals that VEGF signaling mediates the response to neoadjuvant anlotinib combined with PD-1 blockade therapy in non-small cell lung cancer
Ziqi Huang, Li Li, Xiaohe Zhao, Haixia Jin, Meng Shen, Baihui Li, Yu Zeng, Qinfen Zhang, Qiyu Wang, Meng Wang, Lili Yang
Abstract
Clinical trials have shown that neoadjuvant anlotinib combined with PD-1 blockade therapy can prolong the survival of patients with driver gene negative non-small cell lung cancer (NSCLC), but some patients fail to benefit from the combination therapy. To explore the potential drug resistance mechanism and predict the efficacy of neoadjuvant therapy in NSCLC patients, we used scRNA-seq to observe and analyze the dynamic changes of immune cells, stromal cells and cancer cells in NSCLC patients who received neoadjuvant combination therapy. We analyzed transcriptome data of ~ 47,000 single cells from 9 NSCLC patients, including 3 treatment naïve patients, 3 post-treatment patients with major pathological response (MPR), and 3 Non-MPR patients. Subsequently, the infiltration of immune cells was detected by immunohistochemistry and multiplex immunofluorescence in NSCLC. In MPR patients, we found that neoadjuvant therapy reduced the expression of the T cell exhausted signature, reduced the transition of T_THEMIS cells to Tregs, and enhanced the positive feedback between CD4+ T cells and PAX5+ memory B cells. In Non-MPR patients, tumor-associated macrophages (TAMs) dampen therapeutic efficiency by being the hub of cell communication. TAMs and fibroblasts stimulate endothelial cells via VEGF, endothelial ZEB1 may up-regulate FLT1 (VEGFR) expression in response to anlotinib, and VEGFR+ endothelial cell signature can predict survival of NSCLC cohort in TCGA. In addition, PLA2G4A, the key enzyme in the VEGF pathway, was highly expressed in the tumor cells of Non-MPR patients after anlotinib treatment. In 135 NSCLC patients, we confirmed by immunohistochemistry that PLA2G4A was positively correlated with poor prognosis and Tregs infiltration. In conclusion, VEGF signaling dependent dynamic changes in endothelial and epithelial cells are deeply involved in the formation of anlotinib resistance and immunosuppression phenotypes in NSCLC patients.